Name: polyethyleneimine-coated iron oxide nanoparticles as a vehicle for the delivery of small interfering rna to macrophages in vitro and in vivo
Uploaded: 2019-02-05T13:00:00
Description: Watch this Scientific Journal Video about Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo at JoVE.com

This work was supported by the National Natural Science Foundation of China (81772308) and the National Key Research and Development Program of China (No. 2017YFA0205502).

All methods involving live animals were performed in accordance with the animal care and use guidelines of Southeast University, China.
1. Preparation of PEI-SPIONs
<ol>
	<li>Preparation of oleic acid-modified SPIONs
	<ol>
		<li>Dissolve FeCl3&#8226;6H2O and FeSO4&#8226;7H2O in water under the protection of N2.
		<ol>
			<li>Add 28 g of FeCl3&#8226;6H2O and 20 g of FeSO4&#8226;7H2</s...

<ol>
	<li>Murray, P. J., Wynn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+and+pathogenic+functions+of+macrophage+subsets.">Protective and pathogenic functions of macrophage subsets.</a> Nature Reviews Immunology. 11 (11), 723 (2011).</li><li>Mae&#223;, M. B., Wittig, B., Lorkowski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+transfection+of+human+THP-1+macrophages+by+nucleofection.">Highly efficient transfection of human THP-1 macrophages by nucleofection.</a> Journal of Visualized Experiments. (91), e51960 (2014).</li><li>Zhang, X., Edwards, J. P., Mosser, D. 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A., Langer, R., Anderson, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knocking+down+barriers:+advances+in+siRNA+delivery.">Knocking down barriers: advances in siRNA delivery.</a> Nature Reviews Drug Discovery. 8 (2), 129 (2009).</li><li>Liu, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-Alkyl-PEI-functionalized+iron+oxide+nanoclusters+for+efficient+siRNA+delivery.">N-Alkyl-PEI-functionalized iron oxide nanoclusters for efficient siRNA delivery.</a> Small. 7 (19), 2742-2749 (2011).</li><li>Weissleder, R., Nahrendorf, M., Pittet, M. 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Y., Hogg, M. G., Dordick, J. S., Sharfstein, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+delivery+in+three-dimensional+cell+cultures+by+superparamagnetic+nanoparticles.">Gene delivery in three-dimensional cell cultures by superparamagnetic nanoparticles.</a> ACS Nano. 4 (8), 4733-4743 (2010).</li><li>Moghimi, S. M., Hunter, A. C., Murray, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-circulating+and+target-specific+nanoparticles:+theory+to+practice.">Long-circulating and target-specific nanoparticles: theory to practice.</a> Pharmacological Reviews. 53 (2), 283-318 (2001).</li><li>Harvey, A. E., Smart, J. A., Amis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+spectrophotometric+determination+of+iron+(II)+and+total+iron+with+1,+10-phenanthroline.">Simultaneous spectrophotometric determination of iron (II) and total iron with 1, 10-phenanthroline.</a> Analytical Chemistry. 27 (1), 26-29 (1955).</li><li>Duan, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyethyleneimine-functionalized+iron+oxide+nanoparticles+for+systemic+siRNA+delivery+in+experimental+arthritis.">Polyethyleneimine-functionalized iron oxide nanoparticles for systemic siRNA delivery in experimental arthritis.</a> Nanomedicine. 9 (6), 789-801 (2014).</li><li>Fr&#246;hlich, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+surface+charge+in+cellular+uptake+and+cytotoxicity+of+medical+nanoparticles.">The role of surface charge in cellular uptake and cytotoxicity of medical nanoparticles.</a> International Journal of Nanomedicine. 7, 5577 (2012).</li><li>Wu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra-small+particles+of+iron+oxide+as+peroxidase+for+immunohistochemical+detection.">Ultra-small particles of iron oxide as peroxidase for immunohistochemical detection.</a> Nanotechnology. 22 (22), 225703 (2011).</li><li>Xia, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyethyleneimine+coating+enhances+the+cellular+uptake+of+mesoporous+silica+nanoparticles+and+allows+safe+delivery+of+siRNA+and+DNA+constructs.">Polyethyleneimine coating enhances the cellular uptake of mesoporous silica nanoparticles and allows safe delivery of siRNA and DNA constructs.</a> ACS Nano. 3 (10), 3273-3286 (2009).</li><li>Mocellin, S., Provenzano, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference:+learning+gene+knock-down+from+cell+physiology.">RNA interference: learning gene knock-down from cell physiology.</a> Journal of Translational Medicine. 2 (1), 39 (2004).</li><li>Courties, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=et+al.In+vivo+RNAi-mediated+silencing+of+TAK1+decreases+inflammatory+Th1+and+Th17+cells+through+targeting+of+myeloid+cells.">et al.In vivo RNAi-mediated silencing of TAK1 decreases inflammatory Th1 and Th17 cells through targeting of myeloid cells.</a> Blood. 116 (18), 3505-3516 (2010).</li><li>Zolnik, B. S., Gonzalez-Fernandez, A., Sadrieh, N., Dobrovolskaia, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minireview:+nanoparticles+and+the+immune+system.">Minireview: nanoparticles and the immune system.</a> Endocrinology. 151 (2), 458-465 (2010).</li><li>Mulens-Arias, V., Rojas, J. M., P&#233;rez-Yag&#252;e, S., Morales, M. P., Barber, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyethylenimine-coated+SPIONs+trigger+macrophage+activation+through+TLR-4+signaling+and+ROS+production+and+modulate+podosome+dynamics.">Polyethylenimine-coated SPIONs trigger macrophage activation through TLR-4 signaling and ROS production and modulate podosome dynamics.</a> Biomaterials. 52, 494-506 (2015).</li></ol>

Macrophages are refractory to transfect by commonly used nonviral approaches, such as electroporation, cationic liposomes, and lipid species. Here we described a reliable and efficient method to transfect macrophages with siRNA. Using the present protocol, over 90% of macrophage-like RAW 264.7 cells (Figure 2B)&#160;and rat peritoneal macrophages18 can be transfected with siRNA without significant impairment of the cell viability. This method depends on the delivery p...

Macrophages are a type of innate immune cells distributed in all body tissues, albeit in different amounts. By producing a variety of cytokines and other mediators, they play critical roles in the host defense against invading microbial pathogens, in tissue repair following injury, and in maintaining tissue homeostasis1. Due to their importance, macrophages have continuously been the subject of intensive research. However, despite its prevalence in gene regulation and function studies, siRNA-mediated gene silencing is less likely to succeed in macrophages because these cells&#8212;particularly, primary macrophages&#8212;are often difficult to transfect. This can be ascribed to a relatively high degree of toxicity associated with most well-established transfection approaches in which the cell membrane is chemically (e.g., with polymers and lipids) or physically (e.g., by electroporation and gene guns) disrupted to let siRNA molecules cross the membrane, thereby drastically reducing macrophages&#39; viability2,3. Furthermore, macrophages are dedicated phagocytes rich in degradative enzymes. These enzymes can damage the integrity of siRNA, weakening its silencing efficiency even if gene-specific siRNA has been delivered into the cell3,4. Therefore, an effective macrophage-targeted siRNA delivery system needs to protect the integrity and stability of siRNA during delivery4.
It is increasingly evident that dysfunctional macrophages are implicated in the initiation and progression of certain common clinical disorders like autoimmune diseases, atherosclerosis, and cancer. For this reason, modulating macrophage function with, for instance, siRNA, has been emerging as an attractive methodology for treating these disorders5,6,7. Although much progress has been made, a major challenge of siRNA-based treatment strategy is the poor cell specificity of systemically administered siRNA and the insufficient siRNA uptake by macrophages, which consequently lead to undesired side effects. Compared with free nucleic acid therapeutics that usually lack optimal cell selectivity and often lead to off-target adverse effects, drug-loaded nanoparticles (NPs), owing to their spontaneous propensity of being captured by the reticuloendothelial system, can be engineered for passive targeting to macrophages in vivo, allowing for improved therapeutic efficacy with minimal side effects8. Current NPs explored for the delivery of RNA molecules include inorganic nanocarriers, various liposomes, and polymers9. Among them, polyethyleneimine (PEI), a type of cationic polymers capable of binding and condensing nucleic acids into stabilized NPs, shows the highest RNA delivering capacity9,10. PEI protects nucleic acids from enzymatic and nonenzymatic degradation, mediates their transfer across the cell membrane, and promotes their intracellular release. Although initially introduced as a DNA delivery reagent, PEI was subsequently demonstrated to be an attractive platform for in vivo siRNA delivery, either locally or systemically9,10.
Superparamagnetic iron oxide nanoparticles (SPIONs) have shown great promise in biomedicine, owing to their magnetic properties, biocompatibility, comparable size to biologically important objects, high surface-area-to-volume ratio, and easily adaptable surface for bioagent attachment11. For instance, because of their potential utility as a contrast agent and rapid uptake by macrophages, SPIONs have emerged as a favorite clinical tool to image tissue macrophages12. While SPIONs have also been extensively studied as nucleic acid delivery vehicles11,13,14,15, to our knowledge, the literature contains few reports of SPIONs as a carrier for macrophage-targeted siRNA delivery. For gene delivery by SPIONs, their surface is usually coated with a layer of hydrophilic cationic polymers onto which negatively charged nucleic acids can be electrostatically attracted and tethered. Here, we present a method for synthesizing SPIONs whose surface is modified with low-molecular-weight (10 kDa), branched PEI (PEI-SPIONs). These magnetic nanoplatforms are then employed to condense siRNA, forming PEI-SPION/siRNA complexes that enable siRNA transport into the cell. We reason that spontaneous phagocytosis of SPIONs by cells of the reticuloendothelial system16, coupled with the strong ability of binding and condensing nucleic acids by PEI, renders PEI-SPIONs suitable for the efficient transport of siRNA into macrophages. The data presented here support the feasibility of PEI-SPION/siRNA-mediated gene silencing in macrophages in culture as well as in vivo.

<table>
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>C11995500BT</td>
			<td>Warm in 37&#176;C water bath before use</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>A31608-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin (1.5 ml)</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrazolium-based MTS assay kit</td>
			<td>Promega</td>
			<td>G3582</td>
			<td>For cytotoxicity analysis</td>
		</tr>
		<tr>
			<td>RAW 264.7 cell line</td>
			<td>Cell Bank of Chinese Academy of Sciences, Shanghai, China</td>
			<td>TCM13</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture plates (6-well)</td>
			<td>Corning</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture dishes (10 cm)</td>
			<td>Corning</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase-free tubes (1.5 ml)</td>
			<td>AXYGEN</td>
			<td>MCT-150-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes (15 ml)</td>
			<td>Corning</td>
			<td>430791</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Wistar rats</td>
			<td>Shanghai Experimental Animal Center of Chinese 
			Academy of Sciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bacillus Calmette&#8211;Gu&#233;rin freeze-dried powder</td>
			<td>National 
			Institutes for Food and Drug Control, China</td>
			<td></td>
			<td>for inducing adjuvant arthritis in rats</td>
		</tr>
		<tr>
			<td>siRNA</td>
			<td>GenePharma (Shanghai, China)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cy3-siRNA</td>
			<td>RiboBio (Guangzhou, China)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethyleneimine (10 kDa)</td>
			<td>Aladdin Chemical Reagent Co., Ltd.</td>
			<td>E107079</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonia water</td>
			<td>Aladdin Chemical Reagent Co., Ltd.</td>
			<td>A112077</td>
			<td></td>
		</tr>
		<tr>
			<td>Oleic acid</td>
			<td>Aladdin Chemical Reagent Co., Ltd.</td>
			<td>O108484</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethylsulfoxide</td>
			<td>Aladdin Chemical Reagent Co., Ltd.</td>
			<td>D103272</td>
			<td></td>
		</tr>
		<tr>
			<td>FeSO4&#8226;7H2O</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10012118</td>
			<td></td>
		</tr>
		<tr>
			<td>FeCl3&#8226;6H2O</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10011918</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimercaptosuccinic acid</td>
			<td>Aladdin Chemical Reagent Co., Ltd.</td>
			<td>D107254</td>
			<td></td>
		</tr>
		<tr>
			<td>ultrafiltration tube</td>
			<td>Millipore</td>
			<td>UFC910096</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramethylammonium hydroxide solution</td>
			<td>Aladdin Chemical Reagent Co., Ltd.</td>
			<td>T100882</td>
			<td></td>
		</tr>
		<tr>
			<td>Particle size and zeta potential analyzer</td>
			<td>Malvern, England</td>
			<td>Nano ZS90</td>
			<td></td>
		</tr>
	</tbody>
</table>

polyethyleneimine-coated iron oxide nanoparticles as a vehicle for the delivery of small interfering rna to macrophages in vitro and in vivo

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a promising therapeutic target in many disorders, such as autoimmune diseases, atherosclerosis, and cancer. RNAi-mediated gene silencing is a valuable approach of choice to probe and manipulate macrophage function; however, the transfection of macrophages with siRNA is often considered to be technically challenging, and, at present, few methodologies dedicated to the siRNA transfer to macrophages are available. Here, we present a protocol of using polyethyleneimine-coated superparamagnetic iron oxide nanoparticles (PEI-SPIONs) as a vehicle for the targeted delivery of siRNA to macrophages. PEI-SPIONs are capable of binding and completely condensing siRNA when the Fe:siRNA weight ratio reaches 4 and above. In vitro, these nanoparticles can efficiently deliver siRNA into primary macrophages, as well as into the macrophage-like RAW 264.7 cell line, without compromising cell viability at the optimal dose for transfection, and, ultimately, they induce siRNA-mediated target gene silencing. Apart from being used for in vitro siRNA transfection, PEI-SPIONs are also a promising tool for delivering siRNA to macrophages in vivo. In view of its combined features of magnetic property and gene-silencing ability, systemically administered PEI-SPION/siRNA particles are expected not only to modulate macrophage function but also to enable macrophages to be imaged and tracked. In essence, PEI-SPIONs represent a simple, safe, and effective nonviral platform for siRNA delivery to macrophages both in vitro and in vivo.

The size and zeta potential of PEI-SPIONs prepared with this protocol were in the range of 29 - 48 nm (polydispersity index: 0.12 - 0.23) and 30 - 48 mV, respectively. They were stable in water at 4 &#176;C for over 12 months without obvious aggregation. To evaluate their siRNA binding ability, PEI-SPIONs were mixed with siRNA at various Fe:siRNA weight ratios. Figure 1 shows that when the Fe:siRNA weight ratio reaches 4 and above, the band of free siRNA was ...

Watch this Scientific Journal Video about Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo at JoVE.com

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

We describe a method of using polyethyleneimine (PEI)-coated superparamagnetic iron oxide nanoparticles for transfecting macrophages with siRNA. These nanoparticles can efficiently deliver siRNA to macrophages in vitro and in vivo and silence target gene expression.

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a ...

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